Fluorescent compounds have been excellent tools for the sensitive and specific detection of a variety of analytes (De Silva et al., “Signaling Recognition Events with Fluorescent Sensors and Switches,” Chem Rev 97:1515-1566 (1997)). While the rational approach in designing the fluorescent sensors was successful toward diverse small molecule analytes (Gabe et al., “Highly Sensitive Fluorescence Probes for Nitric Oxide Based on Boron Dipyrromethene Chromophore-Rational Design of Potentially Useful Bioimaging Fluorescence Probe,” J Am Chem Soc 126:3357-3367 (2004); Chang et al., “A Selective, Cell-Permeable Optical Probe for Hydrogen Peroxide in Living Cells,” J Am Chem Soc 126:15392-15393 (2004); Burdette et al., “Fluorescent Sensors for Zn(2+) Based on a Fluorescein Platform: Synthesis, Properties and Intracellular Distribution,” J Am Chem Soc 123:7831-7841 (2001); Schneider et al., “Coupling Rational Design with Libraries Leads to the Production of an ATP Selective Chemosensor,” J Am Chem Soc 122:542-543 (2000)), the combinatorial approach to fluorescent dyes has shown powerful advantages owing to a wide range of spectral and structural diversity, developing specific binders for macromolecule structures with a concomitant change of fluorescence properties (Rosania et al., “Combinatorial Approach to Organelle-Targeted Fluorescent Library Based on the Styryl Scaffold,” J Am Chem Soc 125:1130-1131 (2003); Lee et al., “Development of Novel Cell-Permeable DNA Sensitive Dyes Using Combinatorial Synthesis and Cell-Based Screening,” Chem Commun (Camb) 15:1852-1853 (2003); Li et al., “Solid-Phase Synthesis of Styryl Dyes and Their Application as Amyloid Sensors,” Angew Chem Int Ed Engl 43(46):6331-6335 (2004); Li et al., “RNA-Selective, Live Cell Imaging Probes for Studying Nuclear Structure and Function,” Chem Biol 13(6):615-623 (2006)).
The identification of small molecules capable of detecting specific cellular states would facilitate high-throughput screening (Giepmans et al., Science 312:217-224 (2006); Zhang et al., Nat. Rev. Mol. Cell. Biol. 3:906-918 (2002); Finney, N. S., Curr. Opin. Chem. Biol. 10:237-245 (2006)). Many such probes are in current use, but the intended cell state difference is usually within the same cell type, with detection often dependent on either enzymatic or metabolic activity (Smiley et al., Proc. Natl. Acad. Sci. 88:3671-3675 (1991); Pendergrass et al., Cytometry A. 61:162-9 (2004)). A small molecule capable of distinguishing the distinct states resulting from cellular differentiation would be of enormous value, for example, in efforts aimed at regenerative medicine. One example is the use of hydrophobic dyes (Nile Red, Oil Red O) targeting the lipid droplets that accumulate during adipocyte differentiation (Fowler et al., Cytochem. 33:833-836 (1985)). Other cell models of differentiation require more complex measurements of cell state, such as gene expression (Stegmaier et al., Nat. Genet. 36:257-263 (2004); and Hieronymus et al., Cancer Cell. 10:321-330 (2006), which are hereby incorporated by reference in their entirety). Further, efforts toward the identification of fluorescent probes have focused on cell type-specific effects (Sweet et al., Biochem. Biophys. Res. Comm. 314:976-983 (2004)). In the case of myogenesis, a probe capable of distinguishing myoblasts from differentiated myotubes would represent a significant advance over current detection techniques, which typically involve immunofluorescence of proteins expressed selectively in the myotube state, and would facilitate the identification of small molecules involved in the differentiation process.
The present invention is directed to an improved class of fluorescent compounds.